JPH06504561A - Low molecular weight sulfated poly-β-2,6-fructofuranosan and its use for the treatment of osteoarthritis diseases - Google Patents

Abstract

The invention concerns low-molecular sulphated poly- beta -2,6-fructofuranosanes containing monomer units of formula (I), as well as their use for the prophylaxis and treatment of degenerative articular ailments.

Description

[Detailed description of the invention] Low molecular weight sulfated poly-β-2,6-fracdofuranosan and osteoarthritis Its use for treatment Osteoarthritis is a degenerative joint disease with inflammatory symptoms and progressive damage to the cartilage. It is a disease that can lead to functional impairment and even complete reversal. Today this disease The inflammation and pain associated with the disease can be treated, but the progressive damage to the cartilage There are no drugs that have been shown to be able to stop or treat it. deformation Known therapeutic agents for osteoarthritis include, for example, sulfated glucose aminoglycan (Cu rrent Therapeutik Re5earch, 40.6 (10 There are mixtures of 86) 1034) or non-steroidal anti-inflammatory drugs, but these are soft Bone loss cannot be stopped.

Although the pathogenesis of osteoarthritis has not yet been elucidated in detail, chondrocytes are involved in the matrix. Of the major components of this matrix, fermentation It is certain that proteoglycans (PGs) are the first to undergo elementary degradation. It is thought that there is.

Therefore, a promising treatment method for osteoarthritis is based on the action profile of chondrocytes. promotes the synthesis of proteoglycans that promote cartilage damage, and also inhibits the pathological increase in the rate of cartilage damage. This is possible with drugs that stop the disease.

This time, surprisingly, low molecular weight sulfated poly-β-2,6-fracdofuranosan was used as cartilage material. promotes the synthesis of cartilage substances, suppresses damage to cartilage substances, and promotes cartilage proteins. It was found to effectively increase the synthesis of theoglycan. of its pharmacological properties Therefore, these diseases include not only degenerative joint diseases but also rheumatoid arthritis where cartilage destruction is observed. diseases such as rheumatoid arthritis, joint trauma and soft joints after long-term immobilization. It is also highly suitable for the treatment and prevention of osteolysis.

German Patent No. 1 300 668 describes some compounds that can be used in the present invention. However, its use for osteoarthritis is not disclosed.

Therefore, the present invention provides the following formula (■): [Wherein, X is a)-3O3M or b)-(CHz)+m-3OsN (however, M is a physiologically acceptable cation or hydrogen, m is an integer from 2 to 4) and Y is a) hydrogen, b) -5O, M or C) -(C112), -3OjII (just M and m have the meanings given above), and n is an integer from lO to 200. It is a number] sulfated poly-β-2,6-frac having a monomer unit of 1 to 2 and a sulfation degree of 1 to 2. Concerning Dofuranosan.

Preferred compounds of formula I are those where n is an integer from 15 to 75. extremely good Preferred compounds of formula I are those in which n is an integer from 20 to 30.

Examples of suitable physiologically acceptable cations are alkali metals, alkaline earth metals and ammonium cations, including cations of organic ammonium bases. Ru.

The term sulfation degree refers to the sulfo group and testicular unit (fructose) in formula ■. refers to the molar ratio of

The compounds of formula I of the present invention can have a wide range of average molecular weights. Main departure The light compound preferably has an average molecular weight of 3,000 to 65,000, particularly preferably 30 00 to 20,000, most preferably 5,000 to 12,000.

The compounds of the present invention can be used, for example, in poly-β-2,6-fracdofuranosan (levan). with chlorosulfonic acid, preferably in the presence of a base such as pyridine. It can be prepared by The presence of a solvent such as formamide is advantageous. this The process yields levan sulfate in the form of a salt, which is then converted into a suitable In some cases, it is converted into a metal salt, for example a sodium salt, and then subjected to hydrolysis.

Hydrolysis involves adjusting the pal of a solution of a salt of high molecular weight levan sulfate to less than about 2.5. This can be done in a known manner by This is suitable for the solution, i.e. Add a strong acid, such as an inorganic acid such as hydrochloric acid or sulfuric acid, in an amount that satisfies the above p■. This is most easily implemented by adding

Salts of high molecular weight levan sulfate esters contain sulfo group-containing cations in their aqueous solutions. Hydrolysis can be carried out in a particularly simple and gentle manner by adding exchanging agents. this has a similar effect to free strong acids, but compared to the latter, foreign ions are present in solution. It has the advantage of not being intrusive. Suitable ion exchangers are commercially available, e.g. , Product name Amberlite IRC12θ, IRC112, IRC105, D owex 50 or Leuatit KS.

Ion exchange agents can detect the hydrolysis of levan sulfate in a relatively short period of time. They make the solution acidic even before the hydrolysis process is reached. can be removed from the solution after only a short time, e.g. 2-5 minutes, without disturbing the Ru. However, on the other hand, ion exchange agents also slow down the progress of hydrolysis until it is complete. It can also remain in solution undisturbed.

The hydrolysis is carried out at an elevated temperature of 30-70°C, preferably about 50-60°C, at about Conveniently it is carried out for 10 to 100 minutes.

The degree of hydrolysis depends on a decrease in pn, an increase in the hydrolysis time or an increase in the temperature of the solution. It can be made larger, or it can be made smaller by the opposite means.

Levan sulfate having a predetermined molecular weight can be prepared under appropriate conditions within the range stated in the specification. These can be prepared by selecting conditions, and these can be measured in preliminary tests if necessary. .

Levan whose degree of sulfation per sugar unit can be less than 2 is chlorosulfonic acid/levan. It can be prepared by changing the molar ratio. i.e., sulfation per sugar unit, e.g. For levan with a degree of 1.6, choose a chlorosulfonic acid/levan molar ratio of 2.5+1 It can be obtained by

High molecular weight levan can also be prepared with low molecular weight by known methods, e.g. hydrolysis with hydrochloric acid. first converted into quantum levan and then by the known method (DE 1300668) or Sulfation can be carried out by the above method.

The group X and, if appropriate, one of the two groups Y is a sulfatoalkyl group. In order to prepare compounds of the invention, short chain levan fragments are treated with a strong base such as hydroxy For example, 1-chloroethanesulfonate in the presence of sodium hydroxide or potassium hydroxide. halogenoalkylsulfonic acids such as phosphoric acid, or 1,3-propane sultone Alternatively, it is reacted with a sultone such as 1,4-butane sultone.

A preferred embodiment of the process of the invention is to use a solvent at the boiling point temperature of the solvent, such as methanol or in a solution of sodium hydroxide in an alcohol such as ethanol in a molar ratio of 2:1 and involves reacting the sultone with the levan fragment.

The present invention also provides for the prevention and treatment of osteoarthritis, excluding sulfation degree 2. The invention relates to the use of sulfated poly-β-2,6-fracdofuranosan. these are, For example, osteoarthritis, other rheumatic diseases with cartilage damage, rheumatoid arthritis -like arthritis, after joint trauma, for example damage to the meniscus or patella or ligament damage or chondrolysis after long-term joint immobilization. Polysaccharide of the present invention can be administered in a variety of ways. For example, oral, intramuscular, periarticular, intraarticular, intravenous It can be administered internally, intraperitoneally, subcutaneously or rectally.

In addition to conventional pharmacologically acceptable excipients and/or additives, the present invention An effective amount of at least one sulfated poly-β-2,6-fracdofuranosan of the invention It relates to a medicine containing.

The medicament of the present invention comprises at least one sulfated poly-β-2,6-fracdofuranosa into suitable dosage forms, if appropriate with other auxiliaries and/or excipients. Prepare by Auxiliaries and excipients include vehicles, preservatives and other selected from the group consisting of conventional auxiliaries.

For example, for dosage forms for oral administration, starches such as potato starch, corn starch or or starches such as wheat starch, cellulose or its derivatives, especially microcrystalline cellulose. silica, various sugars such as lactose, magnesium carbonate and/or phosphorus. Auxiliary agents such as calcium acids can be used. For example, swelling agents and resins. It is also advantageous to add to the oral dosage form auxiliary agents which improve the tolerance of the drug. Endurance To improve sex, the drug can also be administered in the form of enteric-coated capsules. . Furthermore, the ingredients of the dosage form or composite product may contain, if appropriate, e.g. cellulose or or in the form of permeable membranes based on polystyrene resins or ion exchange agents. It may be advantageous to add agents.

The dosage of the drug of the present invention depends on the dosage form of the drug and the patient's condition, weight and type of disease. Which varies depending on various factors.

- The daily dose is about 100 to 3 000 mg, preferably 300 to 1500 mg.

However, higher or lower daily doses may be used in some situations. good. °Administration of the daily dose of the polysaccharide of the invention may be by single administration or in small doses. You may do this several times in different amounts. It is advisable to administer 2 to 4 times per day.

Example Method 1a: a group poly-β-2,6-fracdofuranosan 227 tl (3.3 mol) of chlorosulfonic acid was added to 6'1 Tol of pyridine and It was added dropwise over 2 hours to a vigorously stirred solution of loloform 400w1, and the reaction temperature temperature was maintained below 10°C.

After the formation of the pyridine/sulfur trioxide complex, 800 Jl of formamide was added at 10°C. and the mixture was stirred for an additional 15 minutes.

Levan 1629 was introduced and the viscous reaction mixture was heated at reflux for 15 minutes. sulfur After the oxidation has taken place, the mixture is cooled to 40-45 °C and mixed with methanol 6 000ml was added.

As a result, the pyridinium salt of levansulfonic acid was separated as an oily precipitate. was obtained, which was further diluted with 30 methanol each time twice after removing the supernatant solvent. Washed with 0ml.

For conversion to the disodium salt, the viscous composition is dissolved in 1201)+l of water, The pH was brought to 10 with 50% strength sodium hydroxide solution. Then, the solution was dissolved in methanol. Le 3500mj! When poured into water, sodium salt precipitates out, and after filtration, It was washed again with 2000 ml of methanol. This reprecipitation to improve purity was repeated and finally the product was vacuum dried at 50°C. Yield: 320g (theoretical value 87%); Molecular weight: (C6H@Na101 +5t) n = (366, 2) n; Analysis: S (theoretical value) = 17.51, 3 (actual value) = 17.47 method 1b: Decomposition of highly polymerized di-sulfated levan to obtain compounds of formula ■ where n is 15 to 50 A solution of 100 q of highly polymerized bisulfated levan of Example 1a in 50 b/m of water was added to Approximately 6.4 g of erlite IRCl2O cation exchange agent was used to obtain a pH of 2.5. p After H stabilized, the exchanged resin was removed by suction filtration, and the filtrate was washed in a water bath with stirring for 6 hours. Heated at 0°C.

The heating time in this case determines the degree of decomposition (see Table 1), and the progress of levan decomposition. The line was checked using a viscometer. After the required viscosity is achieved, the solution is cooled and diluted with 50% concentrated water. Adjust the pH to 6.5-6.8 with sodium oxide solution and concentrate under vacuum to 1 volume. I made it eight. The solution was then poured into 3000 mJ of methanol and added with saturated sodium chloride. 30 ml of lithium solution were added. The formed precipitate is precipitated again in the same way and filtered with suction. and dried under vacuum at 50°C. The results are shown in Table 1.

Table 1 Poly-β-2,6-fracdofuranosan disulfide Example number Decomposition time (min) Viscosity (η average molecular weight 1 30 0.143 20000 2 70 0.093 15000 3 90 0.071 10000 4 120 0.060 8000 5 170 0.053 6500 *) Viscosity measurements (Ubbelohde viscometer) were performed using a 3% aqueous solution at 22°C. did.

Method 2a: A sulfated poly- β-2,6-Frucdofuranosan In a method similar to method 1a), chlorosulfur The resin obtained by selecting the fonic acid/levan molar ratio to be 2.5:1. Ban has a total oxidation degree of 1.6 per testis, and for this purpose, Compared to method 1a), 166 ml of chlorosulfonic acid were required. Yield: 248 f (75% of the theoretical value); Analysis: S (theoretical value) = 14.99%: S (actual value) =14.77% Method 2b= The decomposition of the highly sulfated levan prepared in method 2a) was carried out as described in method 1b). It was carried out. The low molecular weight fractions obtained from this with n of 30 to 60 are listed in Table 2. do.

Table 2 6 11 0.135.20000 7 13 0.078 12000 8 20 0.069 10000 Method 3a: monosulfated poly-β-2,6-fracdofuranosa of formula I, in which group X is a NaO,S group hmm In a method similar to method 1a), monosulfated levan is combined with chlorosulfonic acid versus levan. obtained using a molar ratio of 2:1.

For this purpose, compared to example 1a), chlorosulfonic acid 13:3 wj! (2 mo ) was necessary. Yield: 214 g (81% of theory); Molecular weight: (C6H @Na101) n = (264, 2) n; Analysis: S (theoretical value) = 12.14%; S (actual value) = 12.6% Method 3b = The decomposition of the highly polymerized 1-sulfated levan to obtain the compound of formula l was carried out as in method 1b). It was carried out. The low molecular weight fractions obtained from this are shown in Table 3.

9 8 0.318 20000 10 20 0.089 10000 11 30 0.055 8000 Method 4; group Preparation of fonatopropyl)-poly-β-2,6-fracdofuranosan sodium 4 a) Hydrolysis of highly polymerized levan 120 g of levan was added to a solution of 3.6 a of concentrated hydrochloric acid in 60 (1 + j) of water, and at 4.5 h The mixture was stirred at 25°C for a while. The solution was made neutral with 50% strength sodium hydroxide solution and then Pour methanol 700 (bz) into the solution to remove the wax-like precipitate. Filtered with suction and dried in a vacuum oven at 60°C. Yield: 80g, viscosity: [η) = 0.122 (3% solution in water at 22°C) 4b) Di(3-sulfonatopro pill)-poly-β-2,6-fracdofuranosan sodium salt (Example 12) In a solution of 109 (0.25 mol) sodium hydroxide in 800 ml of ethanol, Sequentially, finely powdered levan 20.39 (0,125 mol) of method 4a then 1.3 - 30.59 (0.25 mol) of propane sultone were added. Then add the mixture to 5 o'clock The mixture was heated under reflux for a while until completely dissolved. Precipitate is formed by slow cooling. , which was filtered and washed with methanol.

Concentrate the remaining filtrate until the liquid is about 156s+1 and add vinegar while stirring vigorously. The mixture was poured into 1200 mJ of ethyl acid. Filter the precipitated crystals and vacuum at 60°C. Dry underneath. Yield = 34.3g (61% of theory); Melting point: 164-165°C (Decomposition); Molecular weight: (C+JtoNaiOzSz)n=(450,4)n, Dashi n is 20-30; Analysis: S (theoretical value) = 14.2%; S (actual value) = 14.7% - viscosity + 0.033 (3% aqueous solution at 22°C) of the polysaccharide of the present invention The activity was investigated in the following experiment.

16 Cartilage matrix synthesis promotion activity in chondrocyte culture test Cells: Hyaline cartilage is extracted from freshly delaminated cow ankle joints, and the original matrix is Pronase (Boehringer Iannheis) and Cola Enzymatically digested with Genase (Sigma), chondrocytes were cultured in 24-well culture dishes. , and cultured in 1% low melting point agarose at a density of 4×10′ cells per well.

Medium: ■＾Ms F12 (Biochrom KG, Berlin ) and 10% fetal bovine serum (Boehringer 1Iannhei*). The test substance was dissolved in the medium. Typically added at a concentration of lo-6M, Newly added each time land was exchanged.

Experimental method Two treatments were carried out from the 3rd day to the 10th day of the initial culture, and on the 98th day, 0tC/mA' of Nag3sSO4 (7,4x 10'Bq) in the medium for 24 hours. Added.

Dissociation and extraction of proteoglycans from the agarose layer was performed using 8M guanidinium hydrochloride. and proteinase inhibitor (Sigma) for 24 h at 4°C with shaking. I did it.

The supernatant after centrifugation was filtered onto a PDIO@Sephadex G25 column to remove free sulfur. The sulfate and incorporated sulfate are separated, and some of their activity is determined by β-scintillation. Measured with a ration counter.

Evaluation: The parameters of matrix production by chondrocytes are The amount of proteoglycan synthesis measured as phase uptake. Each group Nikki The average value is calculated from 4 wells and expressed as a percentage when the untreated control group is taken as 100%. did.

Results: The results are shown in Table 4.

Table 4 = In agarose cell culture ^rteparon book 96-140 cars 4 183-282 5139-184 books 6181~255* 7 125-205 zero 8 177-204 zero 9 Chapters 130-155 10 108~152* 11 108-190 pieces *Average range of several similar experiments Kimoto Lultpold-11erk, 1lunich, FRG2, chondrocyte Cells were prepared and cultured in the same manner as in chondrolytic activity experiment 1 in the culture test.

Medium: From the start of treatment, 5μ/gl of human in complete medium as in test 1. Recombinant interleukin-1α (IL-1, Sigma) was further added; was newly added in the same way as the test substance every time the medium was replaced.

Experimental method Two treatments were carried out at the 5th to 138th stages of the initial culture, and at the 128th stage, the 24 Nat”SOn 201C/ml was added to the culture medium for a period of time. Dissociative extraction and measurement of sulfate uptake were carried out in the same manner as in Test 1.

Evaluation: IL-1 suppresses proteoglycan synthesis and promotes degradation , this is counteracted by the low content of labeled matrix molecules in the agarose layer. It was being shown. Percentages were calculated as in Test 1.

Results: The results are shown in Table 5.

Table 5: In agarose cell culture IL-1 control group 65 IL-1+＾rteparon 103IL-1+4 150 IL-1+ 5 169 IL-1+6 99 IL-1+ 7 189 IL-1+ 8 158 IL-1+ 9 124 IL-1+10 126 IL-1+11 126 IL-1+ 12 88 3. Activity of promoting cartilage synthesis in cartilage transplant culture Cartilage tissue: Standardized discs of hyaline cartilage were punched out from newly delaminated bovine ankle joints. The cells were then cultured in a 24-well culture dish at approximately 200 exposures per well.

Medium: Complete medium containing ■＾Ms F12 and 10% fetal bovine serum, The material was dissolved in the medium. It is normally added at a concentration of 104M and freshly added every time the medium is changed. Added more.

Experimental method: The treatment was carried out from the 3rd day to the 10th day of explant culture, and on the 98th day, 2 0 tC/ml (DNa!”SO4) was added to the medium for 24 hours.

Dissociative and subsequent associative extraction of proteoglycans from cartilage explants was performed using 8M Rapid freezing and thawing using guanidinium hydrochloride and proteinase inhibitors I did this several times. The supernatant after centrifugation was transferred to PDIO Sephadex G. 25 column to separate free sulfate and incorporated sulfate, and Activity was measured in aliquots using a β-scintillation counter.

Evaluation: The parameters of matrix production by chondrocytes are The amount of proteoglycan synthesis measured as phase uptake. for each group The average value is calculated from 6 wells and expressed as a percentage when the untreated control group is taken as 100%. did.

Results: The results are shown in Table 6.

Surface = Professional in cartilage explant culture ^rteparon 121 Submission of translation of written amendment (Article 184-8 of the Patent Law) July 29, 1993

Claims (8)

[Claims] 1. The following formula I: ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) [In the formula, X is a) -SO3M or b )-(CH2)m-SO3M (where M is a physiologically acceptable cation or hydrogen, m is an integer from 2 to 4), Y is a) hydrogen, b) -SO3M or c) -(CH2)m-SO3M (where M and m have the meanings given above), and n is an integer from 10 to 200] sulfated poly-β-2,6-frac having a monomer unit of 1 to 2 and a sulfation degree of 1 to 2. Tofuranosan. 2. The sulfated poly-β-2,6-furan according to claim 1, wherein n is an integer of 15 to 75. Cutofuranosan. 3. The sulfated poly-β-2 according to claim 1 or 2, wherein n is an integer of 20 to 30. 6-Fructofuranosan. 4. Besides conventional pharmaceutically acceptable excipients and/or additives, claim 1 At least one sulfated poly-β-2,6- according to one or more of 3 to 3. A drug containing an effective amount of fructofuranosan. 5. In addition to customary pharmaceutically acceptable excipients and/or additives, the degree of sulfation one or more of the compounds according to claims 1 to 3, not excluding the polysaccharides of claim 2; A drug for the prevention and treatment of degenerative joint diseases containing an effective amount. 6. Degree of sulfation for preparing drugs for the prevention and treatment of osteoarthritis diseases The use of a compound according to one or more of claims 1 to 3, which does not exclude the polysaccharides of claim 2. for. 7. Prevention of degenerative joint disease comprising administering the medicament according to claim 5 and Method of treatment. 8. According to one or more of claims 1 to 3, which does not exclude polysaccharides with a degree of sulfation of 2. one or more substituted polysaccharides together with suitable auxiliaries and/or excipients. for the prevention and treatment of osteoarthritis, including converting it into a suitable dosage form. Method of preparing drugs for.